Optimizing fermentation conditions for fructosyltransferase enzyme production by Lactobacillus plantarum

Enzymatic production of fructooligosaccharides (FOS) from sucrose using β-D fructosyltransferase is commonly employed in commercial scale. FOS are low calorie sweetners with prebiotic property widely used as functional food material. In the present study, a strain of Lactobacillus plantarum (LABF 16) was found to produce bioactive oligosugars such as kestose and nystose exhibiting fructosyltransferase (FTase) enzyme activity. Further maximization of FTase production by the particular isolate was also attempted. The fermentation parameters viz., pH, temperature and sucrose concentration, which was found to influence the fructosyltransferase yield, were optimized by response surface methodology. The optimized conditions for the FTase production were pH 6.33, temperature of 32°C, sucrose concentration of 20.74 g/l, which resulted in an enzymatic activity of 129.62 U/ml/min.


INTRODUCTION
Bioactive oligosaccharides are gaining outstanding popularity because of their excellent functionalities such as being low calorific, non-carcinogenicity and acting as a growth factor for useful microorganism in the intestinal microflora (Nadeau, 2000;Urgell and Orleans, 2001;Patel and Goyal, 2010).Among commercially available dietary sugars, fructooligosaccharides (FOS) possess extraordinary importance as functional food ingredients owing to their prebiotic properties by beneficially affecting the health of the host by selectively stimulating the growth and/or activity of certain gastrointestinal bacteria.Furthermore, their sweet taste is similar to that of sucrose, a traditional sweetener (Huebner et al., 2007).The health benefits associated with FOS includes activation of the human immune system, resistance to infection, enhanced mineral absorption in the gastrointestinal tract, synthesis of B-complex vitamins, lowering of serum cholesterol, suppressing prevalence to diarrhea and preventing carcinogenic tumours (Cummings and Roberfroid, 1997).FOS has received Generally Recorded As Safe (GRAS) status from the Food and Drug Administration (FDA)-U.S. (Godshall, 2007).
Recent research efforts have focused on optimization of process for improved FTase enzyme production.Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of various factors and searching for the optimum conditions.RSM has been successfully used in the optimization of media components in various bioprocesses such as of protease (Adinarayana and Ellaiah, 2002), xylanase (Park et al., 2002), mycophenolic acid (Sadhukhan et al., 1999) and lactic acid production (Hujanen et al., 2001).
The present study was aimed to identify the influential fermentative parameters to increase fructosyltransferase enzyme production by Lactobacillus plantarum (LABF 16) isolated from fermented maize product.RSM was used for further optimization of influential process variables to enhance the yield of enzyme.

Oligosaccharide identification
For the detection of oligosaccharides, the end products of enzyme reaction (1 µl) were spotted on a thin layer chromatography (TLC) ready foil.The TLC foils were run in choloroform : acetic acid : water (6:7:1) and the sugars were specifically stained with diphenyl amine (1.8% w/v) and aniline in phosphoric acid.Sigma grade 1-kestose (GF 2 ), 1-nystose (GF 3 ), glucose and sucrose were used as reference sugars.

Assay for fructosyltransferase
The reaction mixture for the assay of FTase activity consisted of 1.5 ml of 60% (w/v) sucrose in 0.1 M citrate buffer (pH 5.5) and 0.5 ml crude enzyme-culture fluid.The reaction was carried out at 55 ± 1°C for 1 h using a water bath.The reaction was terminated by keeping the reaction mixture in boiling water bath for 15 min.Glucose released at the end of the reaction was estimated using dinitrosalicylic acid (DNS) method (Debois et al., 1956).One unit of FTase activity was defined as the amount of enzyme required to release 1 µmol of glucose released per ml per minute under the above mentioned reaction conditions (Park et al., 2001;Sangeetha et al., 2004).

Effect of different sucrose concentration on FTase production
Sucrose was added to the MRS broth in the range of 10 to 60 g/l.Culture was inoculated and incubated for 48 h at 32°C, 200 rpm in a rotary shaking incubator.The cell was removed from culture broth by centrifugation at 6000 rpm for 10 min and the cell-free culture fluid serve as a crude enzyme source.The enzyme activity was determined by dinitrosalicylic acid (DNS) method (Debois et al., 1956).

Effect of pH and temperature on FTase production
Fifty milliliters of MRS broth containing optimized concentration of sucrose was prepared in 250 ml Erlenmeyer flasks.The pH of MRS media was adjusted to values ranging from 4 to 7 with 4 N NaOH or HCl.The optimum temperature was determined by incubating the inoculated flasks at temperature range from 28-37°C for 48 h.FTase activity was determined by the procedure as described earlier.

Experimental design and statistical analysis
A set of 20 experiments was performed to optimize fermentative parameters for FTase production.Three independent variables, including sucrose concentration, temperature and pH were studied at five different levels (Table 1).One of the response surface methodologies, central composite design was employed for experimental design (Design Expert version 7.0, State-Ease inc., Minneapolis, U.S.A.).The regression and graphical analysis of the data was obtained.The variables were coded according to the Equation 1: (1) Where, x i = Dimensionless value of independent variables, X i = real value of independent variable, = real value of the independent variable at the centre point, Δx 3 = the step change.
The behavior of the system was explained by following the second degree polynomial Equation 2: Y=b 0 +b 1 X 1 +b 2 X 2 +b 3 X 3 +b 11 X 12 +b 22 X 22 +b 33 X 32 +b 12 X 1 X 2 +b 23 X 2 X 3 +b 13 X 1 X 3 (2) Where, Y is predicted response, X 1 , X 2 , X

RESULTS AND DISCUSSION
Plant associated lactic acid bacteria produce a large   Where, x 1, x 2 , x 3 are the coded values of sucrose concentration, temperature, initial pH of medium respectively.Analysis of variance for the selected model is shown in Table 3.The coefficient of correlation (R) was 0.9733 which indicates a good agreement between experimental and predicted values.The coefficient of determination (R 2 =0.9474) is also very high, making the model very significant.The parameter estimated and the corresponding p values (Table 4) suggest that sucrose concentration and temperature are the main factors affecting FTase production.
Results obtained from experimental design showed that increased sucrose concentration was the main consideration of process parameters.This is in agreement with many reports for transferase activity from filamentous fungi (Rehm et al., 1998), yeasts (Boon et al., 2000) and bacteria (Rabiu et al., 2001).The strong effect of substrate concentration observed in our experiments suggested that FTase production could be substantially increased by increasing the sucrose concentration.Moreover, transfructosylating activity was exhibited only under higher sucrose concentration in Aspergillus sp.(Chien et al., 2001;Cuervo et al., 2004).While the strain of L. plantarum (LABF 16) used in our study exhibited fructosyltransferse activity only upto 25 g/l of sucrose concentration.The concentration of sucrose above 25 g/l resulted in reduction of enzyme production.Lower cell growth observed for this culture when it is grown in the MRS medium containing more than 25 g/l sucrose might have resulted in lowered enzyme production for this culture.
The initial pH of medium plays a key role in enzyme production and in utilization of media constituents and growth of the microorganism (Kim et al., 2000;Bonnin and Thibault, 1996).Although we did not find pH as main effect, it was involved in one significant interaction.To keep both pH and temperature at high levels was influential to the enzyme production.In addition, the positive effect of sucrose concentration on the FTase production could be further enhanced by increased temperature due to the interaction between sucrose concentration and temperature (Figure 5).By adopting the RSM, the optimal set of conditions for maximum FTase production was as follows: sucrose concentration of 20.74 g/l, pH 6.33 and temperature of 32°C.At the optimal conditions, a maximum FTase activity of 129.62 U/ml was obtained in batch fermentation process (Table 5).
3 are independent variables, b o is offset term, b 1 , b 2 , b 3 are linear effect, b 11 , b 23 , b 33 are squared effects and b 12 , b 13 , b 33 are interaction terms.

Figure 5 .
Figure 5. Isoresponse contour plots showing the effect of sucrose concentration, pH and temperature on fructosyltransferase enzyme production by L. plantarum (LABF 16)

Table 1 .
Range of values for the response surface method.
*Corresponding author.E-mail: vijiladauphin@yahoo.co.in.Tel: +919443816872.Fax: +914226611294.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Table 3 .
Analysis of variance (ANOVA) for the selected model.

Table 4 .
The least-squares fit and the parameter estimates (significant of regression coefficient).
ACKNOWLEDGEMENTThis work was supported by ICAR Finance Scheme on Application of Microorganisms in Agriculture and Allied Sciences (AMAAS), Ministry of Agriculture, Government of India.REFERENCESAdinarayana K, Ellaiah P (2002).